Cytokines mediate their effects by binding to specific receptors on their target cells. This interaction results in structural changes/modifications in the receptor's cytoplasmic part that eventually trigger intracellular signaling cascades. These signaling cascades usually include enzymes such as kinases, lipases or proteases that mediate the biochemical and genetic changes that are the basis for the biological response to a given cytokine.
As demonstrated for many cytokine receptors—such as the TNF receptor family—one of the first events after ligand binding to a receptor is the recruitment of various adaptor proteins to the signaling domain of the activated receptor (reviewed by Wallach et al. 1999 and Inoue et al. 2000). In a second step, these adaptor proteins mediate specific binding and activation of key enzymes constituting the various signaling cascades. The resulting multi-protein complex consisting of the receptor, and one or various signaling proteins was dubbed “signalosome”. The composition of the signalosome essentially determines which combination of signal pathways is activated in the target cell. The signalosome may therefore be regarded as the central molecular switchboard that determines the eventual biological response to a cytokine.
In view of this molecular mechanism of cytokine receptor signal transduction, it becomes very important to understand the signalosome composition for a given receptor. Knowing the identity of the bound adaptor proteins, the circumstances under which they bind as well as their binding kinetics would allow predictions on the signals initiated by the receptor of interest. Such knowledge would make it also possible to relate signal-cascades or even combinations thereof to biological responses.
CD40, a member of the TNF receptor (TNFR) family, is recognized as the central switch point for the initiation of the interaction between antigen presenting cells (APC) and T-lymphocytes. By influencing basic functions such as cell proliferation, activation and apoptosis this receptor coordinates T-cell priming, the selection process for antibody producing B-cells as well as the activation of the effector mechanisms in macrophages to eliminate intracellular pathogens (reviewed in Grewal et al. 1998, Schonbeck et al. 2001). The importance of CD40 for the maturation and activation of diverse immune effector mechanisms becomes most obvious when this receptor or its ligand fail to function. In mammals this results in a condition known as Hyper IgM syndrome. This severe immune deficiency is characterized by high levels of plasma IgM and low levels of IgA, IgG, and IgE, the absence of germinal centers and the inability to mount a thymus dependent immune response (reviewed in Grewal et al. 1998, Ramesh et al. 1994). Further in vivo studies demonstrated that the CD40-CD40L receptor-ligand pair plays a primary role in the regulation of B-cell proliferation, immunoglobulin production, Ig class switching, rescue of B-cells from apoptosis, germinal center formation, the generation of B-cell memory and the regulation of inflammation (reviewed in Grewal et al. 1998, Schonbeck et al. 2001, Clark et al. 1996 and Foy et al. 1996). CD40's eminent role for the immune response led to the development of various therapeutic concepts in which unwanted immune reactions may be artificially suppressed (reviewed in Grewal et al. 1998 and Liu et al. 1996). It was found that the blockade of CD40-CD40L interactions might be useful for the treatment of autoimmune conditions, transplant rejection, graft versus host reaction and arteriosclerosis (Grewal et al. 1998 and Schonbeck et al. 2001). CD40 belongs to the group of TNF receptors that do not have a ‘death domain’. They generate their signal through direct interaction with TNF receptor associated factors (TRAF) (reviewed in Arch et al. 1998 and Inoue et al. 2000).
TRAFs are a genetically conserved family of proteins that act as adaptors to recruit further signaling molecules such as the kinase NIK (Malinin et al. 1997), thereby activating important downstream effectors such as AP-1 and NF-κB. These transcription factors in turn have been shown to regulate numerous genes involved in various aspects of cellular and immune functions (reviewed in Baeurle et al. 1994, Ghosh et al. 1998 and Karin et al. 1997). Six TRAF proteins are known to date. The first two TRAF1 and TRAF2 were isolated as p75TNF receptor binding proteins (Rothe et al. 1994). All TRAF proteins share a common structural organization. A C-terminal interaction domain, dubbed TRAF domain, mediates the recruitment to various receptors of the TNFR family (Rothe et al. 1995). This domain also mediates homo and hetero-oligomerization and binding to other signaling molecules such as the death domain protein TRADD (reviewed in Arch et al. 1998 and Inoue et al. 2000). The TRAF domain is also the region of highest homology between the different TRAF family members. All TRAFs except TRAF1 have up to 6 repetitive Zn-finger motifs and one ring-finger motif in the N-terminal part that are essential for the activation of downstream components in the signaling pathway such as NIK or JNK (Malinin et al. 1997, Takeuchi et al. 1996, Liu et al. 1996 and Song et al. 1997). In addition TRAF3 and TRAF5 contain a coiled coil structure, which allows homo- and heteromerization between these two TRAFs (Pullen et al. 1998 and Leo et al 1999). Recent crystallization studies have demonstrated that TRAFs interact with their receptors as trimers (McWhirter et al. 1999, Park et al. 1999, Ye et al. 1999 and Ni et al. 2000). The recruitment is most likely achieved by ligand induced receptor trimerization, which results in the approximation of possible interaction sites and thus forming an optimal binding site for the trimeric TRAFs (Baud et al. 1999, Pullen et al. 1999). In view of the multitude of receptors that generate their signals through TRAF proteins it is not surprising that the disruption of TRAF genes in vivo has dramatic consequences. In mice TRAF2-, TRAF3- and TRAF6-deficiencies are lethal (Yeh et al. 1997, Nguyen et al. 1999, Xu et al. 1996, Lomaga et al. 1999 and Naito et al. 1999). A TRAF5 deficiency results in signaling defects for multiple receptors including CD27, CD30, CD40 and the LTJ3 receptor (Nakano et al. 1999). TRAF4 deficient mice are born with a tracheal malformation but no other obvious defects (Shiels et al. 2000). TRAF1 gene targeted mice have not been described.
Despite its relatively short signaling domain of 62 amino acids, CD40 was shown to interact with all TRAFs except TRAF4 (Pullen et al. 1998, Hu et al. 1994, Rothe et al. 1995, Mosialos et al. 1995, Cheng et al. 1995, Ishida et al. b 1996, Ishida et al. a 1996 and Krajewska et al. 1998). On the basis of sequence comparison between TRAF binding receptors two amino-acids sequence motifs were defined as minimal TRAF binding sites: PxQxT for TRAF1, 2 and 3 (Pullen et al. 1998, Cheng et al. 1996, Gedrich et al. 1996, Devergne et al. 1996, Boucher et al. 1997, Eliopoulos et al. 1997 Sandberg et al. 1997, Brodeur et al 1997 and Pullen et al. 1999) and basic-QxPxEx-acidic for TRAF6 (Pullen et al. 1999, Tsukamoto et al. 1999 and Darnay et al. 1999). TRAF5 appears to bind indirectly via TRAF3 (Pullen et al. 1999, Leo et al. 1999). Further refinement of the TRAF2 binding motif to the minimal consensus sequence P/S/T/AxQ/EE was achieved by crystallization studies (Ye et al. 1999). Both TRAF binding motifs are present in CD40 and have been shown to mediate TRAF recruitment to this receptor. Despite extensive structural studies on the CD40-TRAF interactions some basic questions concerning the assembly of the CD40-TRAF signalosome remain unresolved. It is still unclear whether and how the sequence context of the full length CD40 cytoplasmic domain influences the function of the two known TRAF binding motifs. It is also unknown whether the various components in the signalosome mutually affect binding to the receptor, for example by providing additional binding sites for the other signalosome proteins. Resolving these questions will not only be important in order to understand the signaling mechanisms of TRAF binding receptors but also for the rational design of drugs that interfere specifically with TRAF mediated signaling.
A protein designated NIK, including isoforms, analogs, fragments or derivatives thereof which are capable of binding to the tumor necrosis factor receptor-associated (TRAF) proteins is known (Malinin et al. 1997). As the TRAF proteins are involved in the modulation of mediation of the activation of the transcription factor NF-κB, which is initiated by some of the TNF/NGF receptors, as well as others, NIK and its isoforms etc. by binding to TRAF proteins is therefore capable of affecting (modulating or mediating) the intracellular signaling processes initiated by various ligands (e.g. TNF and others) binding to their receptors such as, for example, their modulation/mediation of NF-κB activation, via interaction directly or indirectly with TRAF proteins.
The interaction of cytokine receptors with their signal adaptor proteins was well studied for the TNF receptor gene family. Most assay systems use peptide fragments of the signaling domain of the receptor of interest and test the interaction of this peptide with known adaptor molecules under in-vitro conditions (Rothe et al. 1994, Hu et al. 1994 Boldin et al. 1995, Stanger et al 1995, Chinnaiyan, A. M. et al. 1995, Cheng, G. et al. 1995, Mosialos et al. 1995, Ishida, T et al. a, 1996, Ishida, T. b 1996 and Fields, S. 1989). The systems used comprise the yeast-two-hybrid-system (Fields et al. 1989), immuno-precipitation methods e.g. with Glutathion-S-transferase-tagged receptor fragments (Rothe at all. 1994, Boldin et al. 1995 Chinnaiyan et al. 1995 and Mosialos et al. 1995) or binding of labeled adaptor proteins to peptide receptor fragments spotted on filters (Boucher et al. 1997, Pullen et al. 1998; and Pullen et al. 1999). A common problem of all these methods is that the signalosome assembly occurs not in its natural “juxta-membrane” cellular environment.
A need therefore exists for a simple quantitative assay in which the signalosome assembly occurs in its natural cellular environment as opposed to conventional in vitro conditions.
Immunologic methods for quantifying antigens provide excellent sensitivity and specificity and have become standard techniques for both research and clinical applications. All modern immunochemical methods of protein quantitation are based upon a simple and accurate method for measuring the quantity of an indicator molecule (antigen or antibody) that binds to solid surfaces, such as plastics, and by washing away indicators not bound.
When the indicator molecule is labeled with a radioisotope, the assay is called a radioimmunoassay. The indicator molecule is quantified by counting radioactive decay events in a scintillation counter. The assay is called an Enzyme Linked Immunoadsorbent Assay (ELISA), when the indicator molecule is covalently coupled to an enzyme which can cleave a reporter substrate, which may be colorimetric, chemiluminescent, fluorometric, or phosphometric. The indicator molecule may be quantified by determining with a spectrophotometer the initial rate at which the enzyme converts a neutral substrate to a colored or emitting product.
ELISAs may be classified under four headings: direct, indirect, sandwich and competitive (Crowther, J. R. (1995) Methods in Molecular Biology volume 42 pages 35-50). In the direct-labelled antigen ELISA, the antibodies are adsorbed to the solid-phase and the antigen is labelled. In the direct-labelled antibody ELISA, the antigen that is attached to the solid phase is reacted directly with an enzyme labelled antibody (e.g. conjugated with an enzyme). In the indirect ELISA, the antibody is not labelled and a second antispecies specific antibody conjugated to an enzyme is used.
In the direct-sandwich ELISA, first antibody is attached to the solid phase, the tested antigen can be added and captured by the attached antibody. A second different antibody, conjugated to an enzyme is used to detect the captured antigen. In the indirect-sandwich ELISA the second antibody is not labeled, it is generated in different animal species than the first one, and it is detected by a third antispecies specific labeled antibody.
Competitive ELISA consists of two reactants, which are competing for a third one. The following are examples of competitive ELISAs:
In the direct labelled-antibody-competitive ELISA, the antigen is adsorbed to the solid phase and a pre-titrated conjugated antibody is added, so that the antigen is saturated and no free recognition sites are available for further antibody combination. The interaction of antigen and conjugated antibody is perturbed if the labelled antibody is mixed with another antibody (competing antibody) that is able to react with the solid phase-bound antigen. Such an assay can be used to compare monoclonal antibodies directed against the same protein.
In the direct-antigen-competitive ELISA, the antigen is adsorbed to the solid phase and a pre-titrated conjugated antibody is added so that the antigen is saturated and no free antigenic sites are available for further antibody combination. In this case the interaction of antigen and conjugated antibody is perturbed if the labelled antibody is mixed in with another antigen (competitor). Thus, if the competitor antigen is cross-reactive, the labelled antibody is unavailable to react with the antigen attached to the solid phase, and a reduction in the colour is observed. Such assays are used to quantify antigens or to compare the relative affinity of binding of two antigens for the same antibody.
In the indirect-antigen/antibody-competitive ELISA, the antibody is not labelled and it is detected by a third anti species-specific labelled antibody.
Currently no general, sensitive, specific, easy to perform and efficient method exists which allows quantitation of recruitment of various adaptor proteins to the signaling domain of the activated receptor in intact cells. Therefore the immunologic method described in the present invention solves a long-standing problem in the area of signal transduction.